The Caliciviridae are a family of positive-strand RNA viruses and consist of four genera designated: (1) Norovirus (with species Norwalk virus) (2) Sapovirus (with species Sapporo virus); (3) Vesivirus (with species, feline calicivirus and vesicular exanthema of swine virus); and (4) Lagovirus (with species rabbit hemorrhagic disease virus and European brown hare syndrome virus). Norwalk virus (NV) is the prototype strain for the genus Norovirus and was discovered by LID researchers in 1972. The Noroviruses are the major cause of nonbacterial epidemic gastroenteritis that occurs in family, school, institutional, or community-wide outbreaks, affecting all age groups. The Noroviruses are genetically diverse and cannot be grown in cell culture, which has been a continuing research obstacle. This year, we focused on the molecular characterization of the MD145 strain of the Noroviruses, because our study of the etiology of gastroenteritis outbreaks in Maryland nursing homes had identified this strain as the predominant pathogen. This virus, belonging to the GII/4 genetic cluster of the Noroviruses, has also been identified as the predominant strain associated with calicivirus diarrhea worldwide. A full-length cDNA "consensus clone" of the MD145 genome was constructed, and the proteolytic cleavage map of the nonstructural polyprotein encoded in ORF1 was determined. RNA transcripts derived from the MD145 cDNA clone in the MVA/T7 expression system were not infectious in various cell lines (in contrast to our success with such experiments using a full-length cDNA clone of feline calicivirus) and studies are underway to identify the mechanism responsible for the block in replication. We also attempted the in vivo introduction of capped RNA from our "consensus clone" of the Norwalk virus genome into the intestine of a chimpanzee (animal protocol LID 15), but we did not recover virus. In tandem with these studies of the Noroviruses, we have begun a major new project in the laboratory focusing on the only available enteric calicivirus (porcine enteric calicivirus or PEC) that replicates in cell culture. The PEC will grow in pig kidney cells, but only in the presence of intestinal content fluid from an uninfected gnotobiotic piglet. We are currently working to develop reagents to study viral replication in cells such as an infectious cDNA clone and region-specific antisera. We are working with the NIAID protein support laboratories (including the Bio-Analytical Mass Spectrometry, Peptide Analysis, and Protein Sequencing Units) to characterize the factor(s) in the intestinal content fluid critical for the growth of the PEC in cultured cells. Work continued this year with feline calicivirus (FCV), our model for calicivirus replication. The complete nonstructural polyprotein cleavage map was published and our reverse genetics system allowed the identification of the cleavage sites essential for virus growth and replication. We also reported the first isolation of enzymatically-active membranous replication complexes from feline calicivirus-infected cells.